Pharmacological and computational evaluation of Sapodilla and its constituents for therapeutic potential in hyperactive gastrointestinal disorders

Objective(s): This study was designed to investigate various gastrointestinal effects of Manilkara zapota (Sapodilla), exploring its anti-diarrheal, anti-secretary, anti-spasmodic, anti-ulcer and anti-motility potential. Materials and Methods: Antidiarrheal and anti-secretary activities were investigated using castor oil induced diarrhea and castor oil induced fluid accumulation. Isolated rabbit jejunum tissues (antispasmodic) were employed for in vitro experiments. Antiulcer, antimotility and molecular docking were performed using ethanol-HCl induced ulcer assay, charcoal meal transit time and Auto Doc Vina. Results: Mz.Cr exhibited protection against castor oil-induced diarrhea (P<0.05 vs. saline group) and dose-dependently inhibited intestinal fluid secretions (P<0.001 vs. castor oil group). Mz.Cr caused relaxation of spontaneous and K+ (80 Mm)-induced contractions with EC50 values of 0.11mg/ml (0.08-0.1, n=4) and 0.16 mg/ml (0.09-0.2, n=4) respectively (*P<0.05**P<0.01 ***P<0.001). It showed protective effect against gastric ulcers induced by ethanol-HCl (P<0.001 vs. saline group). Mz.Cr reduced distance travelled by charcoal meal (P<0.001 vs. saline group). Plant constituents: caffeoylquinic acid and methyl 4-O-galloylchlorogenate showed high binding affinities (E-value≥-6.5 Kcal/mol) against histaminergic H2 receptors, H+/K+ ATPase pump and voltage gated L-type calcium channels, while possesses moderate affinities (E-value≥8 Kcal/mol) against histaminergic H1, muscarinic M1, M3 and mu-opioid, whereas lower affinities (E-value≥9.5 Kcal/mol) vs. calmodulin, adrenergic α1, phosphodiesterase enzyme and dopaminergic D2 receptors. Lupeol-3-acetate and β-amyrin-3-(3’-dimethyl) butyrate observed weak affinities. Conclusion: In present study, M. zapota is reported to exhibits anti-diarrheal, anti-secretory, anti-spasmodic, anti-motility, anti-ulcer effects and computational binding affinities against gastrointestinal targets.


Introduction
Gastrointestinal ailments are very common among the people of Asia and medical practitioners believe that it is a root cause for the occurrence of several other co-morbidities. Modern day medicine has so far does not produced any efficacious remedial drug against gastrointestinal disorders. It only gives temporary relief but with side effects. However, traditional herbal medicines have got excellent economical and long lasting potential to treat digestive system disorders (1). These natural products have been a significant source and major contributor to the present day commercial medicines and several drug lead molecules. About 61% of drugs introduced worldwide are derived from natural products (2). Screening of crude plant extracts ease the way for discovery of novel bioactive compounds and their structure elucidation can open the window for new synthetic preparations. For particular therapeutic purposes, pure bioactive compounds can be made in suitable dosage form and their accurate doses can be find out (4). Edible fruits being potential sources of functional foods and its phytoconstituents often serves the purpose in treating and curing several chronic diseases. Use of edible fruit extracts have been reported by several researchers for their gastrointestinal activities (5).
Manilkara zapota L. commonly known as "Sapodilla" and locally "Chiku" belongs to the family of Sapotaceae and is an evergreen, depilated tree up to 15 m in height. Asia is a major cultivator of this species, though it is native to Mexico and Central America (6). M. zapota has been used traditionally in fever, hemorrhage, wound healing, ulcer, arthritis, pulmonary diseases, rheumatism, and as antifungal agent (7). Its use as laxative and for treating constipation and diarrhea, further enhance its ethnomedicinal importance. Fruits are used in traditional medicines as anti-oxidant, due to their polyphenolic content (8).

3-(3'-dimethyl) butyrate (10).
In the present study, we report anti-diarrheal, antisecretary, anti-spasmodic, anti-motility and anti-ulcer effects. Extensive folkloric uses and previous studies were used as a baseline data to validate aforementioned ethnomedicinal uses of the plant. Molecular docking of its constituents with known structure is done to find out the potential lead molecule responsible for pharmacological effects. The 2D and 3D structures of the plant constituents: 4-caffeoylquinic acid (cryptochlorogenic acid), lupeol-3-acetate, methyl 4-O-galloylchlorogenate and β -amyrin-3-(3'-dimethyl) butyrate are presented in Figure 1.

Experimental procedures
Superior quality of M. zapota fruit weighing 4 kg was purchased from local market in Feb 2017. Plant was authenticated by a taxonomist Dr. Mushtaq Ahmad, at Department of Plant Sciences, Quaid-a-Azam University, Islamabad. Voucher specimen no. (ISL-B-23) was collected after submitting sample of specimen of these species to the herbarium at same department. The fruit (4 kg) was air-dried, crushed into powdered form and extracted at room temperature with aqueous-methanol (70:30) three times to obtain M. zapota crude extract (Mz.Cr).

Experimental animals and housing conditions
Sprague-Dawley rats (180-220 g), BALB/c mice (25-30 g) and rabbits (1.0-1.2 kg), of either sex were obtained from animal house of the Riphah Institute of Pharmaceutical Sciences (RIPS) Islamabad. The animals were kept in plastic cages at standard temperature (23-25 °C ). They were fed with standard animal feed and tap water ad libitum. Animals were fasted before each experiment for 24 hr. All the animal experimental protocols were approved by Research and Ethics Committee of RIPS (Ref. No. REC/RIPS/2017/008) which were performed in accordance with the guidelines of "Principles of Laboratory Animal care" (11).

Phytochemical analysis
Detection of major secondary metabolites presence such as glycosides, anthraquinones, steroids, flavonoids and tannins was carried out in Mz.Cr according to standard procedure (12) with slight modifications.

Castor oil induced diarrhea
Previously reported method was used for this study (13). All the test animals were fasted for 24 hr prior to commencement of experimentation. The floor of cage was lined with blotting paper in which animals were placed. First group was assigned as negative control group and received normal saline (10 ml/kg) orally, while second group was given with a dose of loperamide hydrochloride (10 mg/kg, p.o.) and assigned as positive control. Third, fourth and fifth groups received 50, 100 and 300 mg/kg body weight of the extract orally respectively. One hr after administration of the respective doses and treatments, all animals received (10 ml/kg, p.o.) of castor oil. Post treatment evaluation was carried out after waiting 4 hr in order to analyze the diarrheal droppings presence, absence of diarrheal droppings was documented as a positive result. Results were analyzed by applying Chi square test.

Assessment of intestinal fluid accumulation
Intestinal fluid accumulation was determined using the method as described previously (14). To study the intestinal fluid accumulation, entero-pooling assay was used. Overnight fasted mice were taken and put into five assigned cages with five mice in each. Group I and II were administered normal saline (10 ml/kg) and castor oil (10 ml/kg, p.o.) respectively. Extract doses of 50, 100 and 300 mg/kg intraperitoneally were given to Group III, IV and V respectively. Standard drug atropine at dose 10 mg/kg was given to last group, 1 hr prior induction with castor oil (10 ml/kg, p.o.). Mice were sacrificed after 30 min, then intestine was removed and weighed. The results were articulated as: (Pi/Pm) x 1000 where, Pi is the weight (g) of the intestine and Pm is the weight (g) of the animal.

Isolated tissue preparation
Rabbits fasted for 24 hr before experiment but they had a free access to water. Jejunal portion was isolated after cervical dislocation of rabbit and washed with Tyrode's solution. Jejunal segment of 2 cm length was suspended in tissue bath containing Tyrode's solution. Temperature of bath was kept at (37 °C ) and proper aeration of 95% O 2 and 5% CO 2 (carbogen) is ensured. An initial load of 1 g was applied to each tissue and was allowed to equilibrate for 30 min before the addition of any drug. Following equilibration period, each preparation was then stabilized with sub-maximal concentration of ACh (0.3 μM) at 3 min interval until constant responses were recorded via a force displacement transducer (model FT-03) coupled with bridge amplifier and power Lab 4/25 data acquisition system connected to computer running Lab-Chart 6 software (AD Instrument, Sydney Australia). The effects of Mz.Cr at doses (0.01-3mg/mL) was recorded as the % change in the voluntary contractions of jejunum (15).

Charcoal meal transit time
Gastrointestinal transit time was estimated utilizing the charcoal meal test (17). Rats were fasted for 24 hr, the test groups received the extracts at 50, 100 and 300 mg/kg body weight doses, where as positive control group received atropine sulfate (0.1 mg/kg, IP), while the negative control group received normal saline (10 ml/kg, p.o.). 30 mins after all treatments, all the animals were sacrificed. The small intestine was excised after which the distance travelled by charcoal meal through the organ was expressed as a percentage of the length of the small intestine according to the following expression. Intestinal transit(%)=(Distance moved by charcoal meal/ total length of intestine) (cm)×100.

Acute toxicity
Mice were divided in 3 groups of 5 mice each. The test was performed using increasing doses of the plant extract (3 and 5 g/kg) given in 10 ml/kg volume. Saline (10 ml/kg, p.o, negative control) was administered to one group. Twenty-four hr post study the mice were observed for mortality (18).

Statistical analysis
Data was expressed as mean±SEM (n=5) and median effective concentrations (EC 50 ) having 95% confidence intervals. Statistical analysis of the results were analyzed using one-way ANOVA followed by post hoc Tukey's test. Chi square test was used in the case of the anti-diarrheal data, where P<0.05 was regarded as significant. Non-linear regression using Graph Pad program (GraphPAD, SanDiego, CA-USA) was used to analyze the concentration-response curves.

Phytochemical profile
Qualitative phytochemical analysis of Mz.Cr showed the presence of flavonoid, phenols, triterpenes, lignin, unsaturated sterols and carbohydrates.

Effect of Mz.Cr on castor-oil induced diarrhea
Mz.Cr exhibited a dose-dependent (50-300 mg/kg) protective effect against castor oil-induced diarrhea in mice. The negative control group (saline treated) did not show any protection against castor oil-induced diarrhea.

Effect of Mz.Cr on charcoal meal transit time
Mz.Cr hinders the charcoal meal to travel through the small intestine in a dose dependent manner. The distance travelled by the saline group was 82.29%.

Acute toxicity
The extract did not show any mortality up to the dose of 5 g/kg.

Docking evaluation
Assessment of E-value is an important contributor which helps in docking evaluation. Apart from this, other contributing factors include hydrogen bonding, pipi bonding and other hydrophobic interactions between ligand-protein complexes. Results of post dock analysis are given in Tables 4-6 and Table 7, showing number and binding residues of hydrogen bonds, pi-pi bonds

Discussion
Based on ethnopharmacological use of M. zapota in hyperactive gut diseases, such as colic and diarrhea, its extract was evaluated for the possible anti-diarrheal, anti-secretory, charcoal meal gastrointestinal motility and anti-ulcer effects in rodents. Isolated intestinal tissue was used for the elucidation of possible underlying mechanism(s) to rationalize aforementioned ethnomedicinal uses of the plant and it was further supported by virtual screening tools.
Mz.Cr showed protective effect against castor oil induced diarrhea, similar to effect produced by loperamide, a standard drug (11). Castor oil induces diarrhea through its active metabolite i.e. ricinoleic acid. It is responsible for causing diarrhea through a series of actions including activation of small intestinal peristaltic activity with reduction of Na + -K + ATPase activity. These changes eventually result in disturbance in the intestinal mucosa, electrolyte permeability, hypersecretion of intestinal contents, and a slogging of the transport time in the intestine (20). Thus, a potential agent may exhibit its anti-diarrheal activity by these mechanisms. Intracellular Ca 2+ levels had a huge impact on secretary functions of the gastrointestinal organs which lead towards consequences such as discharge of gastric acids and intestinal fluid release. This effect might be affected by some drugs that hinder calcium influx (21). Mz.Cr shows protection against castor oil induced intestinal fluid secretions in mice. The antidiarrheal and anti-secretory activities of Mz.Cr might be because of gastrointestinal relaxant component(s) present in the Mz.Cr.
Spontaneous contracting rabbit jejunum preparation is conventionally used to determine the spasmolytic impact, without the utilization of spasmogen (agonist). In jejunum, papaverine (Ca 2+ influx and phosphodiesterase (PDE) inhibitor) and Mz.Cr both possess repressive effect on spontaneous as well as high K + -induced contractions with similar effect, where as verapamil, a specific calcium antagonist have inhibitory effect against the K + -induced contractions. Against spontaneous and K +induced contractions Mz.Cr produces inhibitory pattern like papaverine does, which depicts that plant may be involved in dual mechanism(s) with CCB, in producing relaxation effect, like PDE enzyme(s) inhibition. PDE enzyme inhibitors augment the intracellular level of cyclic AMP which results in relaxation of smooth muscles (22). Traditionally M. zapota is used in colic and diarrhea, which is observed through its anti-diarrheal, anti-secretory, anti-ulcer and anti-spasmodic effects. This is expected as both Ca 2+ antagonists and PDE inhibitors possess an anti-diarrheal, anti-secretory and anti-spasmodic properties (15).
Various aggressive and protective factors play important role in acid release inside gastrointestinal tract. Any imbalance in these factors results in rupturing of mucosal protection and expose gastric lining to gastric acid leading to the sores called ulcers. To explore the anti-ulcer effect of Mz.Cr, ethanol-HCl induced gastric model was used which through variety of mechanisms stimulates ulcers including mucus exhaustion, mucosal damage, release of superoxide anion, hydroperoxide free radicals, all these mechanisms prolonged the tissue oxidative stress and release of inflammatory mediators (16). Marked inhibition on certain ethanol-HCl induced gastric lesions formation as compared to control group showed gastro protective effect of Mz.Cr. The potential of Mz.Cr to produce anti-ulcer effect might be due to its CCB effect, as Ca 2+ antagonist are well known to demonstrate such effects (23). In pathophysiology of gastric ulcers, oxidative stress plays a vital role. Antioxidant and nitric oxide free radical scavenging activity has been reported by M. zapota (6), which may be responsible for its effectiveness as anti-ulcer agent.
In the small intestinal transit test, Mz.Cr produces suppression of the propulsion of charcoal marker at all test doses just like atropine sulphate a standard drug, that has been reported to have anticholinergic effect on intestinal transit (24). A decrease in the motility of gut muscles increases the stay of substances in the intestine, thus allows better water absorption. This finding suggests that Mz.Cr has the ability to influence the peristaltic movement of intestine thereby indicating the presence of an anti-motility activity. It is therefore presumed that the reduction in the intestinal propulsive movement in the charcoal meal model may be due to antispasmodic properties of the Mz.Cr (25).
The observed therapeutic effects of M. zapota may be due to the presence of phytochemicals, tannins and flavonoids, as these phytoconstituents are well known for gastrointestinal effects. Anti-diarrheal, antisecretory, anti-ulcer and anti-spasmodic activities may be due to flavonoids. Beneficial role of tannins in diarrhea cannot be ignored (26).
In acute toxicity testing, the Mz.Cr did not show any mortality up to the maximum dose (5 g/kg) tested, which shows the wide therapeutic range of M. zapota.
Molecular docking is an effective tool for evaluating the affinity of various protein targets that may possibly be associated with the pathophysiology of gastric disorders. The traditionally acclaimed use of M. zapota in the management of gastric related diseases has been supported with scientific evidence using virtual screening tool.
In this study, Auto Dock Vina program was used through PyRx (27). It uses gradient optimization method and it improves accuracy of binding mode predictions. Hydrogen bonding is reported to be significant in formation of ligand protein complex. In this study, we assessed affinity of ligands through E-value and number of hydrogen bonds against protein targets which imparts their influential effect in gastrointestinal diseases. Lower de-solvation energy is an indication of favorable ligand protein complex which is achieved with lower E-values (28). According to certain instances, no of pipi interactions formed by the ligand-target structural complex contributed to increase the stabilization of complex which is comparable to the stable interaction formed by H-bond. Other hydrophobic bonding likewise improves the partiality of ligand's affinity for particular protein target (29). The affinity of ligands for respective targets was assessed on the basis of atomic energy value, hydrogen bonds, pi-pi interactions and hydrophobic bonding.
It is revealed that 4-caffeoylquinic acid and methyl 4-O-galloylchlorogenate showed more affinity than lupeol-3-acetate and β-amyrin-3-(3'-dimethyl) butyrate. Hydrophobic interactions were shown by ligands with high affinity. Essential amino acids of arginine family are important in the binding site which is involved in interactions with all these ligands (32).